Sustained Stimulation of β2AR Inhibits Insulin Signaling in H9C2 Cardiomyoblast Cells Through the PKA-Dependent Signaling Pathway

Jinli Pei; Zhengpan Xiao; Ziyi Guo; Yechun Pei; Shuangshuang Wei; Hao Wu; Dayong Wang

doi:10.2147/DMSO.S268028

Sustained Stimulation of β₂AR Inhibits Insulin Signaling in H9C2 Cardiomyoblast Cells Through the PKA-Dependent Signaling Pathway

Diabetes Metab Syndr Obes. 2020 Oct 21:13:3887-3898. doi: 10.2147/DMSO.S268028. eCollection 2020.

Authors

Jinli Pei^{1

2}, Zhengpan Xiao^{1

2}, Ziyi Guo^{1

2}, Yechun Pei^{1

2}, Shuangshuang Wei^{1

2}, Hao Wu^{1

2}, Dayong Wang^{1

2}

Affiliations

¹ Key Laboratory of Ministry of Education for Tropical Bioresources, Hainan University, Haikou, Hainan 570228, People's Republic of China.
² Laboratory of Biotechnology and Molecular Pharmacology, School of Life and Pharmaceutical Sciences, Hainan University, Haikou, Hainan 570228, People's Republic of China.

Abstract

Introduction: This study aimed to investigate the role of β₂ adrenergic receptor (β₂AR) in insulin signaling transduction in H9C2 cardiomyoblast cells to understand the formation of the β₂AR-insulin receptor (IR) protein complex and its role in insulin-induced Glut4 expression.

Methods: H9C2 cells were treated with various protein inhibitors (CGP, β₁AR inhibitor CGP20712; ICI, β₂AR inhibitor ICI 118,551; PKI, PKA inhibitor myristoylated PKI; PD 0325901, MEK inhibitor; SP600125, JNK inhibitor) with or without insulin or isoproterenol (ISO) before RNA-sequencing (RNA-Seq) and quantitative-PCR (Q-PCR). Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay were carried out to investigate the formation of the β₂AR-IR protein complex. The intracellular concentrations of cAMP in H9C2 cells were tested by high performance liquid chromatography (HPLC) and the phosphorylation of JNK was tested by Western blot.

Results: Gene Ontology (GO) analysis revealed that the most significantly enriched processes in the domain of molecular function (MF) were catalytic activity and binding, whereas in the domain of biological processes (BP) were metabolic process and cellular process. Furthermore, the enriched processes in the domain of cellular components (CC) were cell and cell parts. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the most significant pathways that have been altered included the PI3K-Akt and MAPK signaling pathways. Q-PCR, which was performed to verify the gene expression levels exhibited consistent results. In evaluating the signaling pathways, the sustained stimulation of β₂AR by ISO inhibited insulin signalling, and the effect was primarily through the cAMP-PKA-JNK pathway and MEK/JNK signaling pathway. Yeast two-hybrid, co-immunoprecipitation and His-tag pull-down assay revealed that β₂AR, IR, insulin receptor substrate 1 (IRS1), Grb2-associated binding protein 1 (GAB1) and Grb2 existed in the same protein complex.

Conclusion: The sustained stimulation of β₂AR might inhibit insulin signaling transduction through the cAMP-PKA-JNK and MEK/JNK pathways in H9C2 cells.

Keywords: RNA sequencing; beta adrenergic receptor; insulin receptor; insulin resistance; protein interaction.

Grants and funding

This work was supported by grants from the National Natural Science Foundation of China (31460226 and 31760246) and Natural Science Foundation of Hainan Province (314058) to Dayong Wang.